Systemic Plasma Analysis

Quantitation of low plasma concentrations of the pharmaceutical cabergoline
is performed to demonstrate the sensitivity and selectivity of the TSQ Quantum
mass spectrometer. Samples with analyte concentrations ranging five orders
of magnitude are analyzed to demonstrate precision and accuracy over a linear
dynamic range suitable for pharmacokinetic applications. Analysis of 50
fg of cabergoline on column in minimally treated plasma samples is performed
to demonstrate the sensitivity, ruggedness, and practicality of the bioanalytical
method in a complex matrix.

A variety of analytical methods have been employed
to quantify low levels of cabergoline in plasma; these
include high performance liquid chromatography
(HPLC), radioimmunassay (RIA), and liquid chromatogra
phy
coupled with tandem mass spectrometry
(LC/MS/MS). Techniques utilizing HPLC alone proved
to have insufficient detection limits.[3,4] RIA also does
not have an adequate lower limit of quantitation
(LLOQ) suitable for monitoring cabergoline when
administered at low doses.[1,5] And while recent
LC/MS/MS methods have shown improvement in
sensitivity through the use of selected reaction
monitoring (SRM), they still require large sample
volumes and time-consuming sample preparation.[6,7]
Consequently, these methods either lack the sensitivity,
dynamic range, or practicality required for routine,
high-throughput pharmacokinetic applications.
To assess the feasibility of using the TSQ Quantum to
address these application requirements, cabergoline
was analyzed in the LC/ESI/SRM, unit-resolution
mode. Significant improvement in sensitivity was
demonstrated on the TSQ Quantum compared to the
previous generation triple quadrupole mass spectrometer
from Thermo Finnigan, the TSQ 7000.[811]
Using the TSQ Quantum, low femtogram-levels of
cabergoline were resolved from the complex plasma
matrix. Excellent precision and accuracy were
maintained over five orders of magnitude, demonstrating
a linear dynamic range suitable for real-world,
pharmacokinetic applications.[8,9,12]

Standard and Sample Preparation: Primary stock
solutions of cabergoline and the internal standard
pergolide (Figure 1) were each prepared at a concentration
of 1 mg/mL in methanol and stored at
25 C. A plasma solution was prepared by precipitating
bovine plasma with a 2 volume of acetonitrile.
A 1 g/mL cabergoline plasma standard
was prepared by spiking the precipitated bovine
plasma with the cabergoline stock solution.
Working plasma standards were prepared by sequentially diluting the 1 g/mL cabergoline
plasma standard with the precipitated bovine
plasma solution to yield final concentrations
ranging from 10 pg/mL to 1 g/mL. Prior to
analysis, each standard level was spiked with the pergolide stock solution to produce the fixed
internal standard concentration of 200 ng/mL of
pergolide. The plasma standards were then ready
for direct injection into the HPLCno further
sample clean up was necessary.

Table 1. Precision and accuracy in the LC/ESI/SRM analysis of cabergoline
in plasma under unit resolution conditions. n >=5 samples at each calibration
level.

Discussion

The quantitative LC/ESI/SRM results for cabergoline
on the TSQ Quantum at unit resolution are
summarized in Figures 24 and in Table 1. The
LLOQ observed for cabergoline was 50 fg on
column (5 L injection of 10 pg/mL) which gave
a S/N ratio of 23 (Figure 2). Bioanalytical methods
for the pharmacokinetic analysis of cabergoline
ideally require detection limits of 12 pg/mL in
plasma. This is easily accomplished on the TSQ
Quantum since an LLOQ of 50 fg on column
represents only 5% (50 L) of a 1 mL plasma
sample containing 1 pg of cabergoline. By extrapolation,
it is clear that extremely sensitive limits
of detection (down to the low pg/mL level for
cabergoline) can be achieved with small plasma
sample volumes.

A significant advantage of using smaller plasma volumes is that it lends
itself to improved pharmacokinetic analyses, as replicate and other large
samples-per-subject study designs would not be limited by the plasma volume
requirements of the assay. The ability to easily attain such low detection
limits on the TSQ Quantum with complex plasma samples also negates the need
to develop highly selective sample enrichment procedures to minimize matrix
interferences and to maximize the amount of analyte injected on the column.[67]
The method used here is in marked contrast to a previous method for cabergoline
developed on the older generation TSQ 7000 triple quadrupole mass spectrometer,
which reported an LLOQ value of 1.86 pg/mL (S/N ratio of 1813.1). However,
this LLOQ actually represents ~900 fg of cabergoline on column[6]
rather than the 50 fg on column reported here. Furthermore, in order to
acquire an accurate, reproducible LLOQ for cabergoline on the TSQ 7000,
the assay required use of a 1-mL plasma sample, a complex extraction procedure
including a five-fol
d enrichment of the analyte concentration, and a large
injection volume (150 L) representing 75% of the extracted sample.[6]
In contrast, using the TSQ Quantum, superior cabergoline sensitivity was
achieved with minimally-treated samples and a much smaller injection volume
(5 L). Thus, the detection limit for cabergoline on the TSQ Quantum
is almost 20 times lower than that attained on the TSQ 7000.[6,8,11]
The calibration curve for cabergoline obtained on the TSQ Quantum shows
a linear dynamic range covering 5 orders of magnitude (1 x 105) with a correlation
coefficient of R > 0.999 using a weighting factor of 1/x (Figure 4). Intra-assay
accuracy and precision was evaluated for n >= 5 samples at each calibration
level. The accuracy and precision numbers obtained over this extended linear
dynamic range are shown in Table 1. The LLOQ (50 fg on column) had an accuracy
(%RE) and precision (%CV) of 13.6% and 9.3% respectively. The %RE and %CV
for all the other calibration levels (0.5 pg to 5000 pg on column) ranged
from -7.5% to 1.6% and 0.5 to 8.4%, respectively. The linear dynamic range
for the TSQ Quantum is considerably broader than that achievable on the
TSQ 7000 (which was less than 2 orders of magnitude for this assay).[6,7]
The significance of this extended linear dynamic range is that a single
method can now be developed for cabergoline for application in both low
and high dose pharmacokinetic analyses.

It is typical for the response of older generation
mass spectrometers to be nonlinear and/or for
detector saturation to occur for linear dynamic
ranges as low as 3 orders of magnitude. This is
particularly problematic for assays where drugs
and their metabolites are present in vastly different
concentrations and for the ana
lysis of drug combination
products. Typically, method development is
biased towards optimizing the detection limit for
the lower concentration analyte, sacrificing
detector saturation of the higher concentration
analytes. Since linear range of the high concentration
analyte is compromised, sample dilution and
subsequent analysis becomes unavoidable; this
contributes considerably to overall sample analysis
time and, thus, does not lend itself to highthroughput
applications. Hence, the extended
linear dynamic range of the TSQ Quantum is
extremely beneficial in these situations.

Conclusions

The TSQ Quantum operated at unit resolution shows tremendous utility in
the development of highly sensitive detection methods for ergoline derivatives
such as cabergoline. Sensitivity on the TSQ Quantum is ~20 times better
than that reported on the TSQ 7000 while only requiring a fraction of the
sample previously used. Furthermore, analysis of low plasma concentrations
of cabergoline was performed in a complex, minimally treated plasma matrix.
The broad dynamic range of the TSQ Quantum, coupled with the excellent precision
and accuracy in unit resolution, is of particular importance for applications
where detector saturation is typically encountered. The broad dynamic range,
therefore, allows for easy development of methods suitable for the analysis
of drugs administered at any given dose (low and high), for drugs and their
metabolites, and for drug combination products. For these reasons, the TSQ
Quantum should find widespread use in the development of sensitive, simple,
fast, accurate, and rugged methods for many pharmacokinetic applications.

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